Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

Usefulness of the hypo-osmotic swelling test for evaluation of human sperm fertilization.

We examined spermatozoa from 135 male patients consulting for infertility to evaluate the usefulness of the hypo-osmotic swelling (HOS) test of sperm fertility for the swim-up washing method. HOS test values significantly improved following the treatment of spermatozoa using the swim-up washing method. There were no differences in the rate of sperm motility between the normal group and the oligozoospermic group following treatment, but the HOS test identified significant differences in swelling rates in both groups before and after treatment. The results of the HOS test following swim-up washing were higher during fertilization attempts for 11 cases of successful intra-uterine insemination (IUI) than during unsuccessful IUI attempts; there were no significant differences between the control group and the successful IUI cases. Moreover, using the lower-normal limits for overall sperm swelling (52%) and g-type sperm swelling (30%) obtained from a normal control group, we found that swelling rates were higher when IUI was successful. These findings indicate that the HOS test is an effective measure of sperm fertilizing function when the swim-up washing method is used for sperm treatment. The score of g-type sperm swelling can be used as a substitute for overall sperm swelling.     

Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection

The hypo-osmotic swelling test, originally developed as a diagnosticsperm test, is used to discriminate viable from non-viable spermatozoafor intracytoplasmic sperm injection (ICSI) in cases of completeasthenozoospermia. In the present study, three hypo-osmoticsolutions were compared, i.e. (A) Jeyendran solution containingsodium citrate and fructose; (B) a mixture of 50% culture mediumand 50% milli-Q water; and milli-Q water. While both the percentageof swelling and vitality assessed by eosin Y remained unchangedafter 5-30 min of sperm exposure to solutions A and B, incubationin water for only 5 min was in itself detrimental. Ten frozen-thaweddonor samples and 10 asthenozoospermic patient samples wereexposed to the three solutions for B and C, but only weaklycorrelated for solution A. Percentage viability was furtherassessed by eosin Y and motility of spermatozoa after 2 h and24 h exposure to the three solutions was compared with unexposedcontrol spermatozoa. While a significant decrease in both parameterswas observed for all three solutions in comparison with thecontrol, sperm quality was significantly higher after exposureto solution B than after exposure to solutions A and C. It maybe concluded that solution B (composed of 50% culture mediumand 50% water) is to be preferred for the selection of viableimmotile spermatozoa for ICSI.     

The hypoosmotic swelling test and cryosurvival of human spermatozoa

The hypoosmotic swelling test is a simple laboratory test to evaluate the functional integrity of the membrane of human spermatozoa. This test was performed on 83 samples of human semen before cryopreservation to determine whether it has any predictive value for the cryosurvival of human spermatozoa. Stepwise regression analysis demonstrated that conventional sperm characteristics, including the concentration, motility, normal morphology and viability, of pre-freeze semen samples were of limited value in predicting the cryosurvival of human spermatozoa. Further, the hypoosmotic swelling test results from pre-freeze semen samples did not correlate with the post-thaw motility or the survival rate of spermatozoa after cryopreservation.     

红细胞冻干保存中的渗透性损伤实验研究

红细胞长期保存中保护剂添加、洗涤过程会引入红细胞渗透性损伤.在冻干保存研究中,由于多种保护剂同时使用,保护剂的类型和功能一直是研究的重点,但很少有从渗透性损伤角度分析保存方案合理性的报道.目前相关文献、专利中所用的保护剂总渗透压差别很大,细胞保存后回收率差异也较大.文中用NaCl溶液实验模拟红细胞保存中保护剂添加、洗涤过程.结果表明,选择合适的添加、洗涤方法可以在一定程度上减小渗透损伤.就红细胞而言,1.5Osmol/kg左右是保护剂总渗透压的一个重要阈值,总渗透压低于该阈值时,渗透性损伤较小;高于该阈值时,渗透损伤随着渗透压的增大而迅速增大.所以选择保护剂时,首先应该根据总渗透压来排除渗透压过高的保存方案,否则红细胞在添加和洗涤保护剂时已经损伤很大.该研究对其它细胞长期保存中保护剂的选择也具有参考意义.     

Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods

Previous reports have left unresolved discrepancies between human sperm cryopreservation methods developed using theoretical optimization approaches and those developed empirically. This study was designed to investigate possible reasons for the discrepancies. Human spermatozoa were exposed to 1 mol/l glycerol, 1 mol/l dimethyl sulphoxide (DMSO), 1 mol/l propylene glycol (PG) or 2 mol/l ethylene glycol (EG) at 22, 11 and 0 degrees C, then returned to isosmotic media while changes in cell volume were monitored. Activation energies (E(a)) of the hydraulic conductivity (L(p)) in the presence of cryoprotective agents (CPA) (L(p)(CPA)) were 22.2 (DMSO), 11.9 (glycerol), 15.8 (PG), and 7.8 (EG) kcal/mol. The E(a) values of the membrane permeability to CPA (P(CPA)) were 12.1 (DMSO), 10.4 (glycerol), 8.6 (PG) and 8.0 (EG) kcal/mol. These data indicated that even at low temperatures, EG permeates fastest. The high L(p)(CPA) in the presence of EG and low associated E(a) would allow spermatozoa to remain closer to equilibrium with the extracellular solution during slow cooling in the presence of ice. Collectively, these data suggest that the increase of the E(a) of L(p) in the presence of CPA at low temperature is the likely reason for the observed discrepancy between theoretical predictions of spermatozoa freezing response and empirical data.     

两种冷冻保护剂对形态正常精子百分率影响

目的比较甘油和甘油-卵黄-柠檬酸钠(GYC)两种冷冻保护剂对精子形态影响.方法应用甘油和GYC两种冷冻保护剂(CPM)对精液标本进行速冻和缓慢冷冻保存,应用计算机精液分析仪进行精子形态分析.结果冷冻复温后形态正常精子百分率与冷冻前比较的形态正常精子百分率比较明显下降(P<0.001);速冻与缓慢冷冻法中两种保护剂间形态正常精子百分率比较均无显著性差异(P>0.05). 结论冷冻保存易造成形态正常精子百分率下降,两种保护剂对精子形态没有影响.     

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

红细胞在保护剂添加和洗涤时的非平衡渗透响应实验研究

在红细胞低温保存或冻干保存中，高浓度保护剂的添加和洗涤会使细胞体积收缩或膨胀，造成溶血损失，但其体积并非单调收缩或膨胀到最后平衡体积，而是要经历更严重的体积变化后再趋于平衡。本研究以NaCl溶液来模拟保护剂的添加与洗涤过程．将38种不同浓度的NaC;溶液，以一步直接法、4步等体积法或4步等摩尔浓度变化法添加到红细胞中，测定其溶血率。若以溶血率10％为标准，红细胞所能承受的最小渗透压在161mOsmol／k附近，而最大渗透压与溶液添加方式有关，等体积添加法效果较好（其值约为5680mOsmol／kg）。研究中还将12％、18％NaCl溶液以4步等体积法加到红细胞中，再用生理盐水以三种方式洗涤，发现等摩尔浓度变化法洗涤效果最好；洗涤后溶血率大大增加，说明很多细胞虽然在保护剂添加时未溶血，但膜脆性已改变，难以恢复到等渗时体积。     

Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody

The study was designed in order to investigate the action ofprogesterone on the spontaneous and ionophore-induced humanspermatozoa acrosome reaction in vitro. The principle of theassay system is flow cytometric analysis of CD46 antibody bindingto the inner acrosomal membrane. The technique is a simple andobjective method of analysis, allowing fluorescent analysisof a large segment (5000 spermatozoa) of the spermatozoa populationunder investigation, with concomitant isolation of the livefraction of the spermatozoa population. Four concentrationsof progesterone (1, 25, 50, and 100µg/ml) were examinedfor their effects on spermatozoa capacitated for 4 and 24 h.In addition, motility parameters were examined by the CellSoft2000 automated semen analyser system. Analysis of variance revealedthat progesterone had no effect on either the spontaneous acrosomereaction or the ionophore-induced acrosome reaction at both4 h and 24 h of spermatozoa capacitation times. Further, noeffects on sperm motility parameters or on spermatozoa viabilitycould be attributed to progesterone. We therefore conclude thatprogesterone had no objectively measurable effects on eitherthe sperm acrosome reaction or sperm motility parameters, asmeasured in normal sperm populations.     

Time course of hypo-osmotic swellings of human spermatozoa: evidence of ordered transition between swelling subtypes

The hypo-osmotic swelling test (HOST or HOS test) usually takes into consideration the total HOS response value with no emphasis either on the value of the response subtypes or the response evaluation time. This study investigated the time course of HOS responses and analysed their physiological relevance. Raw semen spermatozoa and Percoll washed spermatozoa were used in the experiment. The morphological changes in the sperm tail were monitored by incubating the spermatozoa in the hypo- osmotic solution for 16 different time periods. The HOS reactive spermatozoa and the type of HOS reaction (swelling subtypes) of the samples subjected to different duration of treatment were identified under a phase contrast microscope. Also the fate of individual spermatozoa in a hypo-osmotic environment were monitored for 30 min. In spermatozoa exposed to a hypo-osmotic solution, the motility lasted usually less than 2 min and motility characteristics were uniquely different from that of the spermatozoa under iso-osmotic conditions. The HOS response development was permanent but the motility loss due to hypo-osmotic shock was reversible up to 1 min of incubation. There was an indication of ordered transition among the HOS swelling subtypes apparently initiating with subtype b destined to c, d, e, f and g. Further, the subtypes a and g showed gradual decrease and increase, respectively, while subtype b showed abrupt initial increase and then gradual decrease. Transition from b to g could be direct or via one or more than one subtypes. Ultrastructure based analysis indicated that HOS response subtypes are the apparent reflection of the differences in the cytoskeletal assembly of the sperm tail and thus may be identifying different physiological variants in the sperm population. These results indicate that shorter incubation is essential to document the kinetics of various HOS responses but the conventional HOS test misses these important HOS features because of lengthy incubation. Since the time course of ordered transition of HOS responses will vary more than the total HOS response in semen of different aetiologies, the importance of HOS response subtypes and response evaluation time should be taken into consideration when applying HOS test.      

Andrology: Relationship between stimulated hyperactivated motility of human spermatozoa and pregnancy rate in donor insemination: a preliminary report

The proportion of spermatozoa exhibiting the vigorous motilitybehaviour termed ’hyperactivation‘ (HA) has beenshown to be increased following removal of seminal plasma andstimulation with chemical agents such as pentoxifylline. Theaim of this study was to examine the relationship between theproportion of HA in cryopreserved semen samples from sperm donorsand the corresponding pregnancy rates achieved by donor insemination.Cryopreserved samples from 20 men were incubated in the presenceor absence of 3 mM pentoxifylline for 1 h and the %HA determinedin each sample. The relationship between pregnancy rate, theproportion of HA spermatozoa in control and pentoxifyllinetreatedgroups and the change in %HA following pentoxifylline treatment(HA) as well as the mean semen characteristics for each donor[sperm count, motility (%), motility index, normal morphology(%), post-thaw motility (%) and post-thaw motility index] wereexamined by logistic regression of the occurrence of clinicalpregnancy with each insemination. Both HA and mean post-thawmotility index were significantly related to pregnancy ratesand together accounted for 64% of the observed variation inpregnancy rates.     

Germ cell-specific decrease of acrosomal proteolytic activity, sperm motility, and number in mitomycin C-treated mice

Assessment of mammalian sperm acrosomal proteolytic activity, sperm motility, and sperm count may be useful for detecting mutagens, carcinogens, developmentally active agents, and antifertility effects. Groups of six albino mice were given a single i.p. injection of 5 mg/kg mitomycin C (MC) or saline. One treated and one control group of mice were killed 1, 3, 5, 7, or 10 weeks later. Sperm extracted from the vasa deferentia at these killing times were derived from cells treated as spermatozoa, spermatids, preleptotene-late spermatogonial cells, spermatogonial cells, and spermatogonial stem cells. In sperm derived from treated preleptotene or spermatogonial cells, the sperm count, sperm motility, and acrosomal proteolytic activity were decreased significantly. Acrosomal proteolytic activity was also decreased in sperm from spermatogonial stem cells. None of these sperm phenotypes were decreased in treated spermatozoa and spermatids. We propose the hypothesis that induced loss of sperm motility and acrosomal proteolytic activity in single spermatozoa derived from MC-treated spermatogonial cells is caused by mutational or developmental effects, whereas in preleptotene-derived and late-spermatogonium-derived sperm similar dysfunction results from developmental effects. Our data support the hypothesis indirectly. Since a low sperm count is correlated with decreased fertility and acrosomal proteolytic activity is essential for penetration of the zona pellucida by the sperm, the presence of these sperm phenotypes may help to detect chemicals with antifertility effects.     

Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.

The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2-), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 +/- 20.9 U/ml (mean +/- SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 +/- 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehyde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (IVF) or artificial insemination.     

Changes in carnitine and acetylcarnitine in human semen during cryopreservation.

L-Carnitine and acetylcarnitine concentrations were determined in spermatozoa and seminal plasma from 15 men, in both fresh ejaculate and frozen-thawed semen with cryoprotective medium. Sperm motility was also evaluated. In fresh samples, the levels of carnitine and acetylcarnitine in seminal plasma were comparable whereas in spermatozoa, acetylcarnitine predominated. Cryopreservation did not change the carnitine and acetylcarnitine levels in seminal plasma nor the carnitine concentration in spermatozoa; by contrast, the acetylcarnitine level in spermatozoa was decreased in 14 cases (110 +/- 8 versus 210 +/- 20 nmol/10(8) cells). This decrease in acetylcarnitine content was greater during semen dilution in cryoprotectant than after the freezing/thawing process. Motility was also decreased in all cases after the freezing/thawing process. These results suggest that acetylcarnitine recovery in spermatozoa is further evidence of the deleterious effect of the cryoprotective medium in the cryopreservation of semen.     

Osmotic agents for peritoneal dialysis

Glucose has more advantages than drawbacks and is now the sole agent used in clinical practice. Yet there is interest in finding a substitute for glucose as an osmotic agent in peritoneal dialysis solution. Work has identified several promising agents such as albumin, amino acids, gelatin and glycerol but it appears that every one of them, including glucose, would be useful for a short-dwell or for a long-dwell exchange but not for both. Some of them, such as albumin and the amino acids, are close to being an ideal osmotic agent but are prohibitively costly to manufacture. We predict that interest in the future will focus on dialysis solutions containing a mixture of osmotic agents. Such a solution would be acceptable for both short and long-dwell exchanges. It will have a sufficiently low concentration of different agents to minimize toxicity and long-term undesirable side effects. We expect that solutions will be available to better meet patients needs in the near future.     

Hyaluronic acid substantially increases the retention of motility in cryopreserved/thawed human spermatozoa

We have demonstrated previously that hyaluronic acid (HA) improves the velocity and the retention of motility in freshly ejaculated human spermatozoa. In the present work, we examined the effect of HA on cryopreserved/ thawed spermatozoa in four paradigms: (i) effect of HA on sperm motility and velocity in semen; (ii) stabilizing effect of HA after 4 h of incubation when the decline of sperm motility is already detectable; (iii) the duration of improved motility after the separation of spermatozoa from HA by Percoll gradient centrifugation; and (iv) motility of sperm cryopreserved in the presence of HA. HA improved the retention of sperm motility in thawed spermatozoa. Indeed, the motility values after 30 h were approximately 100% higher in the HA compared with the control samples. This effect of HA was also evident in the stabilization of spermatozoa with already declining motility. After removal of the HA from the incubation medium, significantly increased motility in the HA-exposed spermatozoa was still detectable for at least 4 h. Cryopreservation of spermatozoa in the presence of HA did not improve the recovery of motility. The data indicate that HA improves the retention of motility of cryopreserved/thawed spermatozoa, even after the removal of HA from the incubation medium. The utilization of HA will probably prove beneficial in assisted reproduction: in intrauterine insemination and in in-vitro fertilization (IVF), the extended sperm motility and velocity will enhance the fertilizing efficiency; in intracytoplasmic sperm injection (ICSI), the improved motility will facilitate the identification of viable spermatozoa. Because HA is a physiological component of the cumulus and of the female and male reproductive tracts, administration of HA should not cause ethical concerns.      

Prognostic value of two putative sperm function tests: hypo-osmotic swelling and bovine sperm mucus penetration test (Penetrak).

The clinical value of two inexpensive and easy to perform sperm function tests (hypo-osmotic swelling and bovine sperm mucus penetration tests) were examined in a prospective study of in-vivo conception rates in 325 couples. Both tests were of no significant value alone or in combination with traditional semen characteristics in predicting pregnancy outcome. The two most predictive factors of conception were the length of infertility and percentage of progressively motile spermatozoa. Our data support the further investigation of sperm function using more sophisticated quantitative tests, specifically the examination of sperm motility.     

Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa

Semen cryopreservation is an important procedure in the treatment of human infertility. However, the ability of spermatozoa to survive freeze/thaw processes varies between patients. Cryopreservation-induced stress may result in membrane injury with consequent loss of sperm motility and viability. We investigated the relationship between the physico-chemical state of the human sperm membranes and their tolerance to cryopreservation. Conventional characteristics of 20 semen samples were analysed before and after cryopreservation as well as their membrane fluidity assessed by measuring the fluorescence polarization anisotropy, which is inversely proportional to the fluidity. Correlation between fluidity and post-thaw recoveries of motile and viable spermatozoa were examined. Results showed that membrane anisotropy markedly varies between patients. In cryopreserved spermatozoa, anisotropy values were significantly higher than in fresh spermatozoa. Furthermore, recovery of motile and viable spermatozoa after freeze/thaw was strongly correlated with anisotropy of fresh spermatozoa (P < 0.05). The higher the membrane fluidity was before freezing, the better was the response of spermatozoa to cryopreservation. The results indicate that the freeze/thaw process results in a rigidifying effect on the sperm membrane and suggest that sperm adaptability to freeze/thaw-induced stress could be dependent on their initial membrane fluidity. The latter finding has practical implications for predicting the response of spermatozoa following freezing and thawing and for improving the recovery of viable spermatozoa.